羽衣甘蓝泛素结合酶ubc7基因分离、原核表达及纯化

为分离羽衣甘蓝(Brassica oleracea var. acephala)自交不亲和系(S13-bS13-b)泛素结合酶7(ubc7)基因，探讨Ubc7重组蛋白的原核表达及纯化。利用CTAB的方法提取羽衣甘蓝柱头总RNA，通过RT-PCR的方法分离ubc7基因，将编码ubc7基因的cDNA序列亚克隆到pET-14b原核表达载体中，将阳性重组表达质粒转化到大肠杆菌表达菌株BL21(DE3)pLysS中，利用IPTG进行诱导表达，并通过Ni2+-NTA树脂进行亲和层析的方法纯化重组蛋白。结果表明，获得的Boubc7含有一个长度为501 bp的开放读码框，编码一个含有166个氨基酸残基的蛋白质。预测BoUbc7的相对分子质量约为18.7 kDa，理论等电点(pI)为5.35。利用IPTG诱导的细菌蛋白中，SDS-PAGE显示出在分子量大小23 kDa处有蛋白特异性的表达，这与预测的His6融合的BoUbc7蛋白分子量相符；利用Ni2+-NTA树脂进行亲和层析纯化后，成功获得了BoUbc7融合蛋白。该试验分离了羽衣甘蓝ubc7基因，并通过原核表达系统获得了BoUbc7重组蛋白，为进一步分析其生物学功能奠定了基础。

Isolation, Prokaryotic Expression and Purification of ubc7 from Stigma of Ornamental Kale (Brassica oleracea var. acephala)

In order to isolate the ubc7 cDNA (Boubc7) from stigma of ornamental kale (Brassica oleracea var. acephala) S13-b homozygotes and obtain the recombinant BoUbc7 fusion protein from E. coli cells. The total RNA was extracted from stigma of ornamental kale using CTAB method. The ubc7 gene was amplified by RT-PCR. The coding region of Boubc7 was inserted into the pET14b and E.coli BL21 (DE3) pLysS cell was transformed pET-14b-BoUbc7. The recombinant BoUbc7 fusion protein was induced by IPTG and purification using Ni2+-NTA resin. Results showed that, the Boubc7 cDNA contained an opening reading frame of 501 bp, encoding a 166 predicted amino acids residue with the predicted molecular mass of 18.7 kDa and the calculated pI of 5.35. SDS-PAGE results showed that the recombinant BoUbc7 fusion protein about 23 kDa was induced by IPTG and purification by affinity chromatography using Ni2+-NTA resin. The Boubc7 gene and recombinant BoUbc7 fusion protein has been obtained, which laid a solid foundation for investigate the biological function of BoUbc7.